Synthesis of sialic acid in plants

ABSTRACT

A method of synthesizing sialic acid in plants, and plants capable of synthesizing sialic acid is provided. Furthermore, a method of producing sialylated protein in a plant is also provided. The method to synthesize sialic acid comprises providing a plant comprising a nucleotide sequence encoding N-acetyl neuraminic acid (Neu5Ac) synthase or Neu5Ac lyase, and expressing the nucleotide sequence thereby synthesizing sialic acid. The plant may also co-express a nucleotide sequence encoding one or more than one of an epimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter and a sialyltransferase.

FIELD OF INVENTION

The present invention relates to the synthesis of sialic acid in plants.Furthermore the present invention provides methods and plants thatproduce sialic acid, and sialylated proteins produced from these plants.

BACKGROUND OF THE INVENTION

Plants are potentially a low cost and contamination safe factory for theproduction of recombinant pharmaceutical proteins. Most of therecombinant proteins produced in plants are indistinguishable from theirmammalian counterparts, as far as the amino acid sequence, conformationand biological activity. Furthermore, mammalian glycoproteins areefficiently glycosylated when they are expressed in transgenic plants.However, plants produce molecules with N-glycans that differ from thosefound on animal glycoproteins (Lerouge et al., 1998). This may limit theuse of plant-made pharmaceuticals since the presence of plant-specificglyco-epitopes on these proteins may elicit immune responses in humans(Bardor et al., 2003) as well as the absence of mammalian-type epitopes,such as sialylated sequences, may induce their fast clearance from theblood stream. As a consequence, controlling the N-glycosylation ofplant-made pharmaceuticals is a prerequisite for their use in humantherapy.

In planta remodelling strategies have recently emerged to obtainplant-derived antibodies with human compatible carbohydrate profiles.Some strategies involved the retention of the plantibodies in theendoplasmic reticulum (Ko et al., 2003; Sriraman et al., 2004, Trigueroet al., 2005), others involved the transformation of plants withmammalian glycosyltransferases. For example, plant N-glycosylation canbe partially humanised by transformation of plant with a humanβ(1,4)-galactosyltransferase (Palacpac et al., 1999; Bakker et al.,2001). Expression of a murine antibody in a transformed plant resultedin the production of a plant-derived antibody harbouring agalactosylation profile similar to the one observed in the correspondingmurine IgG (Bakker et al., 2001).

Mammalian IgGs bear bi-antennary N-glycans on the conserved site ofN-glycosylation located in the Fc domain. These oligosaccharides areweakly sialylated, and the absence of terminal Neu5Ac does not interferewith the antibody function and stability. In contrast, most othercirculatory glycoproteins have sialylated di-, tri or tetra antennaryN-glycans. The presence of terminal sialic acids on these glycans isrequired for numerous biological functions, the first one being thecontrol of the half-life of the protein in the circulatory system. Inthe absence of terminal sialic acids, glycoproteins are detected byhepatic asialoglycoprotein receptors and cleared from the serum,rendering these proteins biologically short-lived and ineffective (Kelmand Schauer, 1997). Therefore, non-sialylated plant-made pharmaceuticalsmay be rapidly eliminated from the blood stream when injected to ahuman, for example, a tobacco-derived Epo was biologically active invitro but non functional in vivo because of its removal from thecirculation before it reached erythropoietic tissues (Matsumoto et al.,1995).

Remodeling of N-glycans linked to plantibodies into human-like N-glycanshas been already partially achieved in plants by expression of a humanβ(1,4)-galactosyltransferase (Palacpac et al., 1999; Bakker et al.,2001), a transferase that uses the endogenous UDP-Gal as co-substrate. Amammalian sialyltransferase has also been introduced in plants anddemonstrated to be functional and correctly targeted to the Golgiapparatus (Wee et al., 1998). However, no sialylation of endogenousoligosaccharides was observed. The occurrence of sialic acids as well asthe sialylation machinery in plants is still a matter of debate.However, Neu5Ac, the major sialic acid present in humans, as well as itsprecursor N-acetylmannosamine (D-ManNAc) do not appear to be synthesisedin plants in detectable amounts (Séveno et al., 2004). As a consequence,the glyco-engineering of plant N-glycans into sialylatedoligosaccharides requires the co-expression of exogenous enzymes able tocatalyse the synthesis, the activation and the transfer in the Golgiapparatus of Neu5Ac.

In mammals and bacteria, anabolism and catabolism of Neu5Ac occursthrough different pathways (Angata and Varki, 2002). Two main classes ofenzymes are required to form Neu5Ac. N-acetylneuraminate lyases (Neu5Aclyase) is involved in the catabolism of sialic acids by catalysing thecleavage of Neu5Ac into N-acetylmannosamine (D-ManNAc) and pyruvate in areversible reaction. At high concentrations of D-ManNAc and pyruvate,the equilibrium can be shifted to the synthesis of Neu5Ac. Coupled to aglucosamine 2-epimerase activity, Neu5Ac lyase from E. coli was used forthe large-scale production of Neu5Ac from D-GlcNAc (Maru et al., 1998).Alternatively, Neu5Ac synthases, such as NeuB, catalyze the condensationof ManNAc onto phosphoenol pyruvate (PEP) and are directly involved inthe biosynthesis of sialic acids (reviewed in Tanner, 2005).

SUMMARY OF THE INVENTION

The present invention relates to the synthesis of sialic acid in plants.Furthermore the present invention provides methods and plants thatproduce sialic acid, and sialylated proteins produced from these plants.

It is an object of the invention to provide an improved method ofproducing sialic acid in a plant.

According to the present invention there is provided method (A) ofsynthesizing sialic acid, for example N-acetyl neuraminic acid (Neu5Ac),comprising,

i) providing a plant comprising a nucleotide sequence encoding Neu5Acsynthase or Neu5Ac lyase, the nucleotide sequence operatively linkedwith a regulatory region that is active in the plant, and

ii) growing the plant and expressing the nucleotide sequence therebysynthesizing the sialic acid.

Furthermore, after the step of growing, the sialic acid may be recoveredfrom the plant. The regulatory region may be selected from the groupconsisting of a constitutive promoter, an inducible promoter, a tissuespecific promoter, and a developmental promoter.

The present invention also pertains to the method defined above (MethodA), wherein in the step of providing, the plant further comprises asecond nucleotide sequence encoding one or more than one of anepimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and a sialyltransferase, operatively linked toone or more than one second regulatory region active within the plant,and the second nucleotide sequence is co-expressed along with theexpression of the nucleotide sequence. Furthermore, the secondregulatory region may be selected from the group consisting of aconstitutive promoter, an inducible promoter, a tissue specificpromoter, and a developmental promoter.

The present invention also pertains to the method as described above(Method A), wherein the nucleotide sequence encoding Neu5Ac synthase orNeu5Ac lyase, the second nucleotide sequence encoding one or more thanone of the epimerase, CMP-Neu5Ac synthase, or CMP-Neu5Ac transporter, orboth the nucleotide sequence, and the second nucleotide sequence iscodon optimized for expression within the plant.

The present invention provides a method (B) of producing a protein ofinterest comprising,

i) providing a plant that expresses one or more than one firstnucleotide sequence encoding Neu5Ac synthase, Neu5Ac lyase, anepimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and a sialyltransferase, and a second nucleotidesequence encoding the protein of interest,

ii) growing the plant and expressing the first and second nucleotidesequences thereby producing the protein of interest, wherein the proteinof interest is sialylated.

Preferably, the protein of interest that is sialylated comprises di, trior tetra antennary N-glycans.

The present invention also pertains to the method as defined above(Method B) wherein the sialylated protein is extracted from the plant.Furthermore, the sialylated protein may be isolated and purified.

The present invention provides a plant, a plant cell, or a seed,comprising a nucleotide sequence encoding Neu5Ac synthase or Neu5Aclyase operatively linked with a regulatory region that is active in theplant. The plant, the plant cell or the seed may further comprise asecond nucleotide sequence encoding one or more than one of anepimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and a sialyltransferase, operatively linked toone or more than one second regulatory region active within the plant.Furthermore, the regulatory region and the second regulatory regions maybe selected from the group consisting of a constitutive promoter, aninducible promoter, a tissue specific promoter, and a developmentalpromoter.

The present invention pertains to the method as described above (MethodB), wherein the one or more than one first nucleotide sequence encodingNeu5Ac synthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, aCMP-Neu5Ac transporter, a galactosyltransferase, a sialyltransferase, asecond nucleotide sequence encoding the protein of interest, or both thefirst nucleotide sequence, and the second nucleotide sequence is codonoptimized for expression within the plant, plant cell, or the seed.

The present invention includes a plant, a plant cell, or a seed,comprising a nucleotide sequence encoding Neu5Ac synthase or Neu5Aclyase operatively linked with a regulatory region that is active in theplant. The plant, the plant cell or the seed may further comprising asecond nucleotide sequence encoding one or more than one of anepimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and a sialyltransferase, operatively linked toone or more than one second regulatory region active within the plant.Furthermore, the nucleotide sequence encoding Neu5Ac synthase or Neu5Aclyase, the second nucleotide sequence encoding one or more than one ofthe epimerase, CMP-Neu5Ac synthase, or CMP-Neu5Ac transporter, or boththe nucleotide sequence, and the second nucleotide sequence is codonoptimized for expression within the plant, plant cell, or the seed.

The present invention also provides to a plant, a plant cell, or a seed,comprising one or more than one first nucleotide sequence encodingNeu5Ac synthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, aCMP-Neu5Ac transporter, a galactosyltransferase, and asialyltransferase, and a second nucleotide sequence encoding a proteinof interest, the first and second nucleotide sequence operatively linkedwith one or more than one regulatory region that is active in the plant.The one or more than one regulatory region may be selected from thegroup consisting of a constitutive promoter, an inducible promoter, atissue specific promoter, and a developmental promoter. Furthermore, theone or more than one first nucleotide sequence encoding Neu5Ac synthase,Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Actransporter, a galactosyltransferase, a sialyltransferase, a secondnucleotide sequence encoding the protein of interest, or both the firstnucleotide sequence, and the second nucleotide sequence is codonoptimized for expression within the plant, plant cell, or the seed.

The present invention also provides a method (Method C) of synthesizingsialic acid comprising, transiently transforming a plant, or a portionof the plant with a nucleotide sequence encoding N-acetyl neuraminicacid (Neu5Ac) synthase or Neu5Ac lyase, the nucleotide sequenceoperatively linked with a regulatory region that is active in the plant,and expressing the nucleotide sequence thereby synthesizing sialic acid.Furthermore, the Neu5Ac or the Neu5Ac lyase may be recovered from theplant or a portion of the plant.

The present invention also pertains to the method as described above(Method C), wherein in the step of transiently transforming the plant ora portion of the plant, further comprises a second nucleotide sequenceencoding one or more than one of an epimerase, a CMP-Neu5Ac synthase, ora CMP-Neu5Ac transporter, operatively linked to one or more than onesecond regulatory region active within the plant, and the secondnucleotide sequence is co-expressed along with the expression of thenucleotide sequence.

The present invention provides a method (method D) of producing aprotein of interest comprising,

i) transiently transforming a plant or portion of the plant with aconstruct that expresses one or more than one first nucleotide sequenceencoding Neu5Ac synthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Acsynthase, a CMP-Neu5Ac transporter, a galactosyltransferase, asialyltransferase, and a second nucleotide sequence encoding the proteinof interest, and

ii) producing the protein of interest, wherein the protein of interestis sialylated.

The protein of interest that is sialylated may comprise di, tri or tetraantennary N-glycans. Furthermore, the sialylated protein may extractedfrom the plant or portion of the plant. The sialylated protein ofinterest may also be isolated and purified.

The present invention also pertain to the method as described above(Method D), wherein after the step of producing, plant materialcomprising the sialylated protein of interest is orally administered toa subject. For example, after the step of producing, the plant orportion of the plant may be minimally processed to produce minimallyprocessed plant material, and the minimally processed plant materialcomprising the sialylated protein of interest orally administered to asubject.

As described herein, the expression in plants of Neu5Ac-synthesisingenzymes, Neu5Ac lyase and NeuB2, results in the accumulation offunctional enzymes within plant tissues, Neu5Ac-synthesising enzymes maybe expressed in any plant, for example but not limited to tobacco andMedicago sativa (alfalfa), the perennial legume crop that benefits fromseveral agronomic advantages for molecular farming applications (Busseet al., 2001).

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1a shows a Western-blot analysis of soluble proteins extracted fromwild-type (line 1) or transgenic tobacco BY2 cells expressing the Neu5Aclyase-FLAG (line 2) using anti-FLAG antibodies. FIG. 1b and FIG. 1c showa Gas chromatography profiles of the end-products obtained afterincubation at pH 7 and 37° C. of cytosolic proteins, isolated fromtobacco BY2 cells expressing the Neu5Ac lyase, without (FIG. 1b ) orwith (FIG. 1c ) Neu5Ac. FIG. 1d shows a GC profile of cytosolicmonosaccharides of tobacco BY2 cells expressing the Neu5Ac lyase fedduring 48 h at 37° C. with exogenous 10 mM Neu5Ac. FIG. 1e and FIG. 1fshow Electron impact mass spectra of the peak 1 (FIG. 1e ), and peaks 2and 3 (FIG. 1f ) detected in profile (FIG. 1c ). Main fragment ions of1-O-methyl persilyl derivatives of D-ManNAc are indicated.

FIG. 2a and FIG. 2b show Gas chromatography profiles of the end-productsobtained after incubation at pH 7 and 37° C. of cytosolic proteins,isolated from tobacco BY2 cells expressing the Neu5Ac lyase, without(FIG. 2a ) or with (FIG. 2b ) D-ManNAc and pyruvate. FIG. 2c showsElectron impact mass spectrum of the peak appearing in profile (FIG. 2b). Main fragment ions of 1-O-methyl methylester persilyl derivatives ofN-acetylneuraminic acid are indicated.

FIG. 3a shows a Western-blot analysis of cytosolic proteins extractedfrom wild-type (line 1) or transgenic tobacco BY2 cells expressing theNeuB2-FLAG (line 2) using anti-FLAG antibodies. FIG. 3b and FIG. 3c showGas chromatography profiles of the end-products obtained afterincubation at pH 8 and 37° C. of soluble proteins, extracted from leavesof alfalfa plants expressing the NeuB2, without (FIG. 3b ) or (FIG. 3c )D-ManNAc and PEP. FIG. 3d shows an Electron impact mass spectrum of thepeak appearing in profile (FIG. 3c ). Main fragment ions of 1-O-methylmethylester persilyl derivatives of N-acetylneuraminic acid areindicated.

DETAILED DESCRIPTION

The present invention relates to the synthesis of sialic acid in plants.Furthermore the present invention provides methods and plants thatexpress sialic acid, and sialylated proteins produced from these plants.

The following description is of a preferred embodiment.

The present invention provides a method for the synthesis N-acetylneuraminic acid (Neu5Ac) within plants. Neu5Ac lyase catabolize sialicacids in bacteria by catalysing the cleavage of Neu5Ac into ManNAc andpyruvate in a reversible reaction. As this reaction is reversible,Neu5Ac lyase may be used to synthesis of Neu5Ac in the presence of theappropriate precursors. An alternate method for the production of Neu5Acinvolves the use of Neu5Ac synthase. Neu5Ac synthase catalyzes theformation of Neu5Ac by condensation of D-ManNAc and PEP.

Therefore, the present invention provides a method of synthesizingNeu5Ac comprising, providing a plant comprising a nucleotide sequenceencoding Neu5Ac synthase or Neu5Ac lyase, the nucleotide sequenceoperatively linked with a regulatory region that is active in the plant,growing the plant, and expressing the nucleotide sequence to synthesizeNeu5Ac. Alternatively, the method may involve transient production ofNeu5Ac within a plant, or a portion of the plant.

The Neu5Ac so produced may be recovered from the plant and used forsialylation of proteins in vitro, using processes known within the art.Alternatively, the Neu5Ac may be used as an endogenous substrate for thesialylation of a protein of interest that is co-expressed within theplant.

If desired, the levels of substrate for the synthesis of Neu5Ac withinthe plant, including but not limited to N-acetylmannosamine (D-ManNAC),may be increased by co-expressing within the plant one or more than oneadditional nucleotide sequence encoding one or more than one of anepimerase, a CMP-Neu5Ac synthase, and a CMP-Neu5Ac transporter. Forexample, ManNAc may be synthesized by expressing within a plant,UDP-GlcNAc 2-epimerase, for example a bacterial UDP-GlcNAc 2-epimerase,or an epimerase form other sources, which converts endogenous UDP-GlcNAcinto ManNAc. Alternatively, ManNAc-6-phosphate may be produced, followedby hydrolysis with a phosphatase. With this approach GlcNAc-6-phosphate2-epimerase, for example a bacterial GlcNAc-6-phosphate 2-epimerase, ora mammalian UDP-GlcNAc 2-epimerase/ManNAc kinase is expressed within aplant. By co-expressing this second nucleotide sequence along with theexpression of the nucleotide sequence encoding Neu5Ac synthase or Neu5Aclyase, increased levels of Neu5Ac may be produced. However, the need forco-expressing one or more of the above nucleotide sequences may dependupon the host plant selected, as endogenous activities of one or more ofthese enzymes may be present within the plant.

To ensure sialylation of N-glycans from cytosolic sialic acid, bacterialor mammalian CMP-Neu5Ac synthase, mammalian CMP-Neu5Ac transporter,mammalian galactosyltransferase, (for the addition of galactose, beforesialic acid can be transferred to N-glycans). and mammaliansialyltransferase may be co-expressed within a plant. Neu5Ac producedwithin the plant according to the present invention may be used as asubstrate for the synthesis of CMP-N-acetylneuraminic acid (CMP-Neu5Ac)via CMP-Neu5Ac synthase, the CMP-Neu5Ac is then used as a substrate forthe sialylation of a protein of interest that is also co-expressedwithin the plant. In this case the plant may also comprise a nucleotidesequence encoding a sialyltransferase. Expression of a mammaliansialyltransferase, and mammalian CMP-Neu5Ac synthase in plants has beendemonstrated (Wee et al., 1998, Misaki, R., et al., 2006, which areincorporated herein by reference). However, the need for co-expressingone or more of the above nucleotide sequences may depend upon the hostplant selected, as endogenous activities of one or more of these enzymesmay be present within the plant.

In the cases where nucleotide sequences are co-expressed within theplant, each of the desired nucleotide sequences may be introduced intothe plant using standard transformation techniques, transienttransformation techniques, or two plants, each expressing one or more ofthe desired nucleotide sequences may be crossed to obtain a plant thatco-expresses the required combination of nucleotide sequences.

Therefore, the present invention also provides a method for producing aplant that may be used as a platform for the production of a sialylatedprotein of interest. This method comprises, providing a plant thatexpresses one or more than one first nucleotide sequence encoding Neu5Acsynthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, aCMP-Neu5Ac transporter, a galactosyltransferase, and asialyltransferase, and expressing the one or more nucleotide sequence.In order to produce the protein of interest, either a second nucleotidesequence encoding the protein of interest is introduced into theplatform plant using standard techniques, for example transformation,and the second nucleotide sequence is expressed, or the platform plantis crossed with a plant expressing the protein of interest so that theprotein of interest produced within the progeny of the crossed plants issialylated.

The present invention also provides a method for producing a protein ofinterest comprising, providing a plant that expresses one or more thanone first nucleotide sequence encoding Neu5Ac synthase, Neu5Ac lyase, anepimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and a sialyltransferase, and a second nucleotidesequence encoding the protein of interest, growing the plant, andexpressing the first and second nucleotide sequences thereby producingthe protein of interest, wherein the protein of interest is sialylated.Preferably, the protein of interest that is sialylated comprises di, trior tetra antennary N-glycans. The sialylated protein may be extractedfrom the plant, and if desired, the sialylated protein may be isolatedand purified using standard methods. Again, the plants may be eitherstably transformed with the desired constructs, or the plant or portionof the plant may be transiently transformed with the desired constructs.

The nucleotide sequences encoding Neu5Ac synthase, Neu5Ac lyase,epimerase, CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and sialyltransferase may be codon optimized toincrease the level of expression within the plant. By codon optimizationit is meant the selection of appropriate DNA nucleotides for thesynthesis of oligonucleotide building blocks, and their subsequentenzymatic assembly, of a structural gene or fragment thereof in order toapproach codon usage within plants.

In order to optimize the expression of the foreign sequence within aplant, the nucleotide sequence, which may be a wild type or syntheticsequence may be used or altered as required so that the correspondingprotein, for example Neu5Ac synthase, Neu5Ac lyase, epimerase,CMP-Neu5Ac synthase, CMP-Neu5Ac transporter, galactosyltransferase,sialyltransferase, the protein of interest, or a combination thereof, isproduced at a level higher than would be produced when encoded by theun-modified nucleotide sequence. For example, which is not to beconsidered limiting, the sequence may be a synthetic sequence, optimizedfor codon usage within a plant, comprising at least about 80% homologywith the wild type sequence, as determined using sequence comparisontechniques for example but not limited to BLAST (available throughGenBank; using default parameters). It is also contemplated thatfragments or portions of the sequence encoding the protein of interest,or derivatives thereof, that exhibit useful biological properties, forexample but not limited to antigenic properties, may be expressed withinplant tissues.

In order to maximize expression levels and transgene protein productionof Neu5Ac synthase, Neu5Ac lyase, epimerase, CMP-Neu5Ac synthase,CMP-Neu5Ac transporter, galactosyltransferase, sialyltransferase, and aprotein of interest, the nucleic acid sequence may be examined and thecoding region modified to optimize for expression of the gene in plants,using a procedure similar to that outlined by Sardana et al. (Plant CellReports 15:677-681; 1996). A table of codon usage from highly expressedgenes of dicotyledonous plants is available from several sourcesincluding Murray et al. (Nuc Acids Res. 17:477-498; 1989).

Therefore, the present invention provides a method of synthesizingsialic acid comprising, providing a plant comprising a nucleotidesequence encoding N-acetyl neuraminic acid (Neu5Ac) synthase or Neu5Aclyase, the nucleotide sequence operatively linked with a regulatoryregion that is active in the plant, growing the plant, and expressingthe nucleotide sequence thereby synthesizing sialic acid. The nucleotidesequence encoding Neu5Ac synthase or Neu5Ac lyase may be codon optimizedfor expression within the plant. Furthermore, in the step of providing,the plant may further comprise a second nucleotide sequence encoding oneor more than one of an epimerase, a CMP-Neu5Ac synthase, or a CMP-Neu5Actransporter, operatively linked to one or more than one secondregulatory region active within the plant, and the second nucleotidesequence is co-expressed along with the expression of the nucleotidesequence. The second nucleotide sequence encoding one or more than oneof the epimerase, CMP-Neu5Ac synthase, or CMP-Neu5Ac transporter, may becodon optimized for expression within the plant.

Additionally, the present invention provides a method of producing aprotein of interest comprising providing a plant that expresses one ormore than one first nucleotide sequence encoding Neu5Ac synthase, Neu5Aclyase, an epimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, a sialyltransferase, and a second nucleotidesequence encoding the protein of interest, growing the plant, andexpressing the first and second nucleotide sequences thereby producingthe protein of interest. The one or more than one first nucleotidesequence encoding Neu5Ac synthase, Neu5Ac lyase, an epimerase, aCMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, a galactosyltransferase,a sialyltransferase, a second nucleotide sequence encoding the proteinof interest, or both the first nucleotide sequence, and the secondnucleotide sequence may be codon optimized for expression within theplant.

Furthermore, the present invention pertains to a plant, a plant cell, ora seed, comprising a nucleotide sequence encoding Neu5Ac synthase orNeu5Ac lyase operatively linked with a regulatory region that is activein the plant. The plant, plant cell, or seed may further comprise asecond nucleotide sequence encoding one or more than one of anepimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Ac transporter, agalactosyltransferase, and a sialyltransferase, operatively linked toone or more than one second regulatory region active within the plant.The nucleotide sequence encoding Neu5Ac synthase or Neu5Ac lyase, thesecond nucleotide sequence encoding one or more than one of theepimerase, CMP-Neu5Ac synthase, or CMP-Neu5Ac transporter, or both thenucleotide sequence and the second nucleotide sequence, may be codonoptimized for expression within the plant, plant cell or plant seed.

The present invention also includes a plant, a plant cell, or a seed,comprising one or more than one first nucleotide sequence encodingNeu5Ac synthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, aCMP-Neu5Ac transporter, a galactosyltransferase, and asialyltransferase, and a second nucleotide sequence encoding a proteinof interest, the first and second nucleotide sequence operatively linkedwith one or more than one regulatory region that is active in the plant.The one or more than one first nucleotide sequence encoding Neu5Acsynthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, aCMP-Neu5Ac transporter, a galactosyltransferase, a sialyltransferase, asecond nucleotide sequence encoding the protein of interest, or both thefirst nucleotide sequence, and the second nucleotide sequence may becodon optimized for expression within the plant.

By “operatively linked” it is meant that the particular sequencesinteract either directly or indirectly to carry out an intendedfunction, such as mediation or modulation of gene expression. Theinteraction of operatively linked sequences may, for example, bemediated by proteins that interact with the operatively linkedsequences. A transcriptional regulatory region and a sequence ofinterest are operably linked when the sequences are functionallyconnected so as to permit transcription of the sequence of interest tobe mediated or modulated by the transcriptional regulatory region.

By the term “plant matter”, it is meant any material derived from aplant. Plant matter may comprise an entire plant, tissue, cells, or anyfraction thereof. Further, plant matter may comprise intracellular plantcomponents, extracellular plant components, liquid or solid extracts ofplants, or a combination thereof. Further, plant matter may compriseplants, plant cells, tissue, a liquid extract, or a combination thereof,from plant leaves, stems, fruit, roots or a combination thereof. Plantmatter may comprise a plant or portion thereof which has not besubjected to any processing steps. However, it is also contemplated thatthe plant material may be subjected to minimal processing steps asdefined below, or more rigorous processing, including partial orsubstantial protein purification using techniques commonly known withinthe art including, but not limited to chromatography, electrophoresisand the like.

By the term “minimal processing” it is meant plant matter, for example,a plant or portion thereof comprising a protein of interest which ispartially purified to yield a plant extract, homogenate, fraction ofplant homogenate or the like. Partial purification may comprise, but isnot limited to disrupting plant cellular structures thereby creating acomposition comprising soluble plant components, and insoluble plantcomponents which may be separated for example, but not limited to, bycentrifugation, filtration or a combination thereof. In this regard,proteins secreted within the extracellular space of leaf or othertissues could be readily obtained using vacuum or centrifugalextraction, or tissues could be extracted under pressure by passagethrough rollers or grinding or the like to squeeze or liberate theprotein free from within the extracellular space. Minimal processingcould also involve preparation of crude extracts of soluble proteins,since these preparations would have negligible contamination fromsecondary plant products. Further, minimal processing may involveaqueous extraction of soluble protein from leaves, followed byprecipitation with any suitable salt. Other methods may include largescale maceration and juice extraction in order to permit the direct useof the extract.

The plant matter, in the form of plant material or tissue may be orallydelivered to a subject. The plant matter may be administered as part ofa dietary supplement, along with other foods, or encapsulated. The plantmatter or tissue may also be concentrated to improve or increasepalatability, or provided along with other materials, ingredients, orpharmaceutical excipients, as required.

It is contemplated that a plant comprising the heterologous protein ofinterest may be administered to a subject, for example an animal orhuman, in a variety of ways depending upon the need and the situation.For example, if the protein is orally administered, the plant tissue maybe harvested and directly feed to the subject, or the harvested tissuemay be dried prior to feeding, or an animal may be permitted to graze onthe plant with no prior harvest taking place. It is also consideredwithin the scope of this invention for the harvested plant tissues to beprovided as a food supplement within animal feed. If the plant tissue isbeing feed to an animal with little or not further processing it ispreferred that the plant tissue being administered is edible.Furthermore, the protein of interest obtained from the plant may beextracted prior to its use as a food supplement, in either a crude,partially purified, or purified form. In this latter case, the proteinmay be produced in either edible or non-edible plants.

As described in more detail in the Examples, Neu5Ac lyase, and Neu5Aclyase-FLAG (Neu5Ac lyase tagged at its C-terminus with a FLAG epitope toallow immunodetection of the recombinant protein in transformants) wereintroduced into plants. Western-blot analysis, using anti-FLAGantibodies, demonstrated that a protein of MW_(r) 32 kDa was present inthe transformed cells (FIG. 1a ). Furthermore both in vitro and in vivolyase activity was detectable in extracts obtained from, or plantsexpressing either Neu5Ac lyase or Neu5Ac lyase-FLAG. No endogenous lyaseactivity was detected in non-transformed plants. However, the synthesisof Neu5Ac using recombinantly produced Neu5Ac lyase, in the presence ofD-ManNAc and pyruvate was observed (see FIG. 2b ). Therefore,recombinantly expressed Neu5Ac lyase is biologically active in planta.

Neu5Ac synthase (for example, but not limited to NeuB2) and Neu5Acsynthase-FLAG (Neu5Ac synthase tagged at its C-terminus with a FLAGepitope to allow immunodetection of the recombinant protein intransformants) were introduced into plants. Western-blot analysis, usinganti-FLAG antibodies, demonstrated that a protein of MW_(r) 37 kDa waspresent in the transformed cells. Both in vitro and in vivo synthaseactivity was detectable in extracts obtained from, or plants expressingeither Neu5Ac synthase or Neu5Ac synthase-FLAG. No endogenous synthaseactivity was detected in non-transformed plants. However, the synthesisof Neu5Ac using recombinantly produced Neu5Ac synthase in the presenceof D-ManNAc and PEP was observed (see FIGS. 3b, 3c ). Therefore,recombinantly expressed Neu5Ac synthase is biologically active inplanta.

An “analogue” or “derivative” includes any substitution, deletion, oraddition to the silencing nucleotide sequence, provided that thenucleotide sequence retains the property of silencing expression of atarget gene or sequence, reducing expression of a target sequence, orreducing synthesis or activity of a protein encoded by the targetsequence. For example, derivatives, and analogues of nucleic acidsequences typically exhibit greater than 80% similarity with, asilencing nucleic acid sequence. Sequence similarity, may be determinedby use of the BLAST algorithm (GenBank:ncbi.nlm.nih.gov/cgi-bin/BLAST/), using default parameters (Program:blastn; Database: nr; Expect 10; filter: low complexity; Alignment:pairwise; Word size: 11). Analogs, or derivatives thereof, also includethose nucleotide sequences that hybridize under stringent hybridizationconditions (see Maniatis et al., in Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory, 1982, p. 387-389, or Ausubel, etal. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., New York, atp. 2.10.3) to any one of the sequences described herein, provided thatthe sequences exhibit the property of silencing expression of a targetgene. An example of one such stringent hybridization conditions may behybridization with a suitable probe, for example but not limited to, a[gama-³²P]dATP labelled probe for 16-20 hrs at 65EC in 7% SDS, 1 mMEDTA, 0.5M Na₂HPO₄, pH 7.2. Followed by washing in 5% SDS, 1 mM EDTA 40mM Na₂HPO₄, pH 7.2 for 30 min followed by washing in 1% SDS, 1 mM EDTA40 mM Na₂HPO₄, pH 7.2 for 30 min. Washing in this buffer may be repeatedto reduce background.

By “regulatory region” “regulatory element” or “promoter” it is meant aportion of nucleic acid typically, but not always, upstream of theprotein coding region of a gene, which may be comprised of either DNA orRNA, or both DNA and RNA. When a regulatory region is active, and inoperative association, or operatively linked, with a gene of interest,this may result in expression of the gene of interest. A regulatoryelement may be capable of mediating organ specificity, or controllingdevelopmental or temporal gene activation. A “regulatory region”includes promoter elements, core promoter elements exhibiting a basalpromoter activity, elements that are inducible in response to anexternal stimulus, elements that mediate promoter activity such asnegative regulatory elements or transcriptional enhancers. “Regulatoryregion”, as used herein, also includes elements that are activefollowing transcription, for example, regulatory elements that modulategene expression such as translational and transcriptional enhancers,translational and transcriptional repressors, upstream activatingsequences, and mRNA instability determinants. Several of these latterelements may be located proximal to the coding region.

In the context of this disclosure, the term “regulatory element” or“regulatory region” typically refers to a sequence of DNA, usually, butnot always, upstream (5′) to the coding sequence of a structural gene,which controls the expression of the coding region by providing therecognition for RNA polymerase and/or other factors required fortranscription to start at a particular site. However, it is to beunderstood that other nucleotide sequences, located within introns, or3′ of the sequence may also contribute to the regulation of expressionof a coding region of interest. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.Most, but not all, eukaryotic promoter elements contain a TATA box, aconserved nucleic acid sequence comprised of adenosine and thymidinenucleotide base pairs usually situated approximately 25 base pairsupstream of a transcriptional start site. A promoter element comprises abasal promoter element, responsible for the initiation of transcription,as well as other regulatory elements (as listed above) that modify geneexpression.

There are several types of regulatory regions, including those that aredevelopmentally regulated, inducible or constitutive. A regulatoryregion that is developmentally regulated, or controls the differentialexpression of a gene under its control, is activated within certainorgans or tissues of an organ at specific times during the developmentof that organ or tissue. However, some regulatory regions that aredevelopmentally regulated may preferentially be active within certainorgans or tissues at specific developmental stages, they may also beactive in a developmentally regulated manner, or at a basal level inother organs or tissues within the plant as well. Examples oftissue-specific regulatory regions, for example see-specific aregulatory region, include the napin promoter, and the cruciferinpromoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau etal., 1994, Plant Cell 14: 125-130).

An inducible regulatory region is one that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically the protein factorthat binds specifically to an inducible regulatory region to activatetranscription may be present in an inactive form, which is then directlyor indirectly converted to the active form by the inducer. However, theprotein factor may also be absent. The inducer can be a chemical agentsuch as a protein, metabolite, growth regulator, herbicide or phenoliccompound or a physiological stress imposed directly by heat, cold, salt,or toxic elements or indirectly through the action of a pathogen ordisease agent such as a virus. A plant cell containing an inducibleregulatory region may be exposed to an inducer by externally applyingthe inducer to the cell or plant such as by spraying, watering, heatingor similar methods. Inducible regulatory elements may be derived fromeither plant or non-plant genes (e.g. Gatz, C. and Lenk, I. R. P., 1998,Trends Plant Sci. 3, 352-358; which is incorporated by reference).Examples, of potential inducible promoters include, but not limited to,tetracycline-inducible promoter (Gatz, C., 1997, Ann. Rev. PlantPhysiol. Plant Mol. Biol. 48, 89-108; which is incorporated byreference), steroid inducible promoter (Aoyama, T. and Chua, N. H.,1997, Plant J. 2, 397-404; which is incorporated by reference) andethanol-inducible promoter (Salter, M. G., et al, 1998, Plant Journal16, 127-132; Caddick, M. X., et al, 1998, Nature Biotech. 16, 177-180,which are incorporated by reference) cytokinin inducible IB6 and CKI1genes (Brandstatter, I. and Kieber, J. J., 1998, Plant Cell 10,1009-1019; Kakimoto, T., 1996, Science 274, 982-985; which areincorporated by reference) and the auxin inducible element, DR5(Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which isincorporated by reference).

A constitutive regulatory region directs the expression of a genethroughout the various parts of a plant and continuously throughoutplant development. Examples of known constitutive regulatory elementsinclude promoters associated with the CaMV 35S transcript. (Odell etal., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991,Plant Cell, 3: 1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121), or tms 2 (U.S. Pat. No. 5,428,147, which is incorporatedherein by reference), and triosephosphate isomerase 1 (Xu et. al., 1994,Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejoet al, 1993, Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1and 6 genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-646), and thetobacco translational initiation factor 4A gene (Mandel et al, 1995Plant Mol. Biol. 29: 995-1004). The term “constitutive” as used hereindoes not necessarily indicate that a gene under control of theconstitutive regulatory region is expressed at the same level in allcell types, but that the gene is expressed in a wide range of cell typeseven though variation in abundance is often observed.

The one or more than one nucleotide sequence of the present inventionmay be expressed in any suitable plant host that is transformed by thenucleotide sequence, or constructs, or vectors of the present invention.Examples of suitable hosts include, but are not limited to, agriculturalcrops including alfalfa, canola, Brassica spp., maize, tobacco, alfalfa,potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, andcotton.

Therefore, the present invention also provides for a plant, a plantcell, or a seed comprising a nucleotide sequence encoding Neu5Acsynthase or Neu5Ac lyase operatively linked with a regulatory regionthat is active in the plant. Furthermore, the plant, the plant cell orthe seed may comprising a second nucleotide sequence encoding one ormore than one of an epimerase, a CMP-Neu5Ac synthase, a CMP-Neu5Actransporter, a galactosyltransferase, and a sialyltransferase,operatively linked to one or more than one second regulatory regionactive within the plant.

The present invention also provides a plant, a plant cell, or a seed,comprising one or more than one first nucleotide sequence encodingNeu5Ac synthase, Neu5Ac lyase, an epimerase, a CMP-Neu5Ac synthase, aCMP-Neu5Ac transporter, a galactosyltransferase, and asialyltransferase, and a second nucleotide sequence encoding a proteinof interest, the first and second nucleotide sequence operatively linkedwith one or more than one regulatory region that is active in the plant.

The one or more chimeric genetic constructs of the present invention canfurther comprise a 3′ untranslated region. A 3′ untranslated regionrefers to that portion of a gene comprising a DNA segment that containsa polyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by effecting the addition of polyadenylic acidtracks to the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon. One or more of thechimeric genetic constructs of the present invention can also includefurther enhancers, either translation or transcription enhancers, as maybe required. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence.

Non-limiting examples of suitable 3′ regions are the 3′ transcribednon-translated regions containing a polyadenylation signal ofAgrobacterium tumor inducing (Ti) plasmid genes, such as the nopalinesynthase (Nos gene) and plant genes such as the soybean storage proteingenes and the small subunit of the ribulose-1,5-bisphosphate carboxylase(ssRUBISCO) gene.

To aid in identification of transformed plant cells, the constructs ofthis invention may be further manipulated to include plant selectablemarkers. Useful selectable markers include enzymes that provide forresistance to chemicals such as an antibiotic for example, gentamycin,hygromycin, kanamycin, or herbicides such as phosphinothrycin,glyphosate, chlorosulfuron, and the like. Similarly, enzymes providingfor production of a compound identifiable by colour change such as GUS(beta-glucuronidase), or luminescence, such as luciferase or GFP, may beused.

Also considered part of this invention are transgenic plants, plantcells or seeds containing the chimeric gene construct of the presentinvention. Methods of regenerating whole plants from plant cells arealso known in the art. In general, transformed plant cells are culturedin an appropriate medium, which may contain selective agents such asantibiotics, where selectable markers are used to facilitateidentification of transformed plant cells. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be used toestablish repetitive generations, either from seeds or using vegetativepropagation techniques. Transgenic plants can also be generated withoutusing tissue cultures.

The regulatory elements of the present invention may also be combinedwith coding region of interest for expression within a range of hostorganisms that are amenable to transformation, or transient expression.Such organisms include, but are not limited to plants, both monocots anddicots, for example but not limited to corn, cereal plants, wheat,barley, oat, tobacco, Brassica, soybean, bean, pea, alfalfa, potato,tomato, ginseng, and Arabidopsis.

Methods for transient expression, transformation, and regeneration ofthese organisms are established in the art and known to one of skill inthe art. The method of obtaining transformed and regenerated plants isnot critical to the present invention.

By “transformation” it is meant the interspecific transfer of geneticinformation that is manifested genotypically, phenotypically, or both.The interspecific transfer of genetic information from a chimericconstruct to a host may be heritable and the transfer of geneticinformation considered stable, or the transfer may be transient and thetransfer of genetic information is not inheritable. The presentinvention further includes a suitable vector comprising the chimericgene construct suitable for use with either stable or transientexpression systems.

The constructs of the present invention can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, micro-injection, electroporation, etc. For reviews ofsuch techniques see for example Weissbach and Weissbach, Methods forPlant Molecular Biology, Academy Press, New York VIII, pp. 421-463(1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); andMiki and Iyer, Fundamentals of Gene Transfer in Plants. In PlantMetabolism, 2d Ed. D T. Dennis, D H Turpin, D D Lefebrve, D B Layzell(eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). Othermethods include direct DNA uptake, the use of liposomes,electroporation, for example using protoplasts, micro-injection,microprojectiles or whiskers, and vacuum infiltration. See, for example,Bilang, et al. (Gene 100: 247-250 (1991), Scheid et al. (Mol. Gen.Genet. 228: 104-112, 1991), Guerche et al. (Plant Science 52: 111-116,1987), Neuhause et al. (Theor. Appl Genet. 75: 30-36, 1987), Klein etal., Nature 327: 70-73 (1987); Howell et al. (Science 208: 1265, 1980),Horsch et al. (Science 227: 1229-1231, 1985), DeBlock et al., PlantPhysiology 91: 694-701, 1989), Methods for Plant Molecular Biology(Weissbach and Weissbach, eds., Academic Press Inc., 1988), Methods inPlant Molecular Biology (Schuler and Zielinski, eds., Academic PressInc., 1989), Liu and Lomonossoff (J Virol Meth, 105:343-348, 2002), U.S.Pat. Nos. 4,945,050; 5,036,006; and 5,100,792, U.S. patent applicationSer. No. 08/438,666, filed May 10, 1995, and Ser. No. 07/951,715, filedSep. 25, 1992, (all of which are hereby incorporated by reference).

As described below, transient expression methods may be used to expressthe constructs of the present invention (see Liu and Lomonossoff, 2002,Journal of Virological Methods, 105:343-348; which is incorporatedherein by reference). These methods may include, for example, but arenot limited to, a method of Agro-inoculation or Agro-infiltration,however, other transient methods may also be used as noted above. Witheither Agro-inoculation or Agro-infiltration, a mixture of Agrobacteriacomprising the desired nucleic acid enter the intercellular spaces ofthe a tissue, for example the leaves, aerial portion of the plant(including stem, leaves and flower), other portion of the plant (stem,root, flower), or the whole plant. After crossing the epidermis theAgrobacteria infect and transfer t-DNA copies into the cells. The t-DNAis episomally transcribed and the mRNA translated, leading to theproduction of the protein of interest in infected cells, however, thepassage oft-DNA inside the nucleus is transient.

By “gene of interest”, “nucleotide sequence of interest”, or “codingregion of interest”, it is meant any gene, nucleotide sequence, orcoding region that is to be expressed within a host organism, forexample a plant. These terms are used interchangeably. Such a nucleotidesequence of interest may include, but is not limited to, a gene orcoding region whose product is an industrial enzyme, a proteinsupplement, a nutraceutical, a value-added product, or a fragmentthereof for feed, food, or both feed and food use. A nucleotidesequence, or coding region of interest may also include a gene thatencodes a pharmaceutically active protein, for example growth factors,growth regulators, antibodies, antigens, and fragments thereof, or theirderivatives useful for immunization or vaccination and the like. Suchproteins include, but are not limited to, interleukins, for example oneor more than one of IL-1 to IL-24, IL-26 and IL-27, cytokines,Erythropoietin (EPO), insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF orcombinations thereof, interferons, for example, interferon-alpha,interferon-beta, interferon-gama, blood clotting factors, for example,Factor VIII, Factor IX, or tPA hGH, receptors, receptor agonists,antibodies, neuropolypeptides, insulin, vaccines, growth factors forexample but not limited to epidermal growth factor, keratinocyte growthfactor, transformation growth factor, growth regulators, antigens,autoantigens, fragments thereof, or combinations thereof.

If the gene of interest encodes a product that is directly or indirectlytoxic to the plant, then by using the method of the present invention,such toxicity may be reduced throughout the plant by selectivelyexpressing the gene of interest within a desired tissue or at a desiredstage of plant development.

The coding region of interest or the nucleotide sequence of interest maybe expressed in any suitable plant host which is either transformed orcomprises the nucleotide sequences, or nucleic acid molecules, orgenetic constructs, or vectors of the present invention. Examples ofsuitable hosts include, but are not limited to, Arabidopsis,agricultural crops including for example canola, Brassica spp., maize,tobacco, alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat,barley, sunflower, and cotton.

Sialic acid synthesis, for example, Neu5Ac synthesis, in plants wasdemonstrated by expressing recombinant Neu5Ac lyase or Neu5Ac synthase.Neu5Ac lyase from E. coli and NeuB2 from C. jejuni were each expressedin the cytosol of tobacco BY2 cells, alfalfa plants byAgrobacterium-mediated transformation, or when transiently expressed inplant cells. No degradation of the recombinant proteins was observedindicating that these enzymes are stable in this compartment. The Neu5Aclyase expressed in BY2 cells was able to catalyse the cleavage of Neu5Acinto D-ManNAc and pyruvate in a reversible reaction. The synthesis ofNeu5Ac in presence of pyruvate and ManNAc was also observed. Neu5Aclyase was biologically active at pH 7 and over a 25-37° C. range whichis consistent with both pH of the plant cytosol and temperature of mostimportant crops. Furthermore, feeding experiments carried out inpresence of exogenous Neu5Ac demonstrated that the enzyme was functionalin planta.

The Neu5Ac synthase, NeuB2 from C. jejuni, when expressed in tobacco BY2cells was observed to synthesize Neu5Ac in presence of D-ManNAc and PEP.Expression of NeuB2 in alfalfa plants also resulted in an accumulationof a functional enzyme. Therefore, expression of a microbial Neu5Aclyase or Neu5Ac synthase in plants results in the production in thecytosol of enzymes able to synthesise Neu5Ac.

An epimerase able to convert the endogenous GlcNAc into ManNAc, may beco-expressed in plants in order to supply Neu5Ac lyase or Neu5Acsynthase with the appropriate aminosugar substrate. In this regard, theexpression of a functional CMP-Neu5Ac synthase and CMP-Neu5Actransporter in tobacco BY2 cells has been reported (Misaki et al.,2006). By co-expressing CMP-Neu5Ac synthase, CMP-Neu5Ac transporter, orboth CMP-Neu5Ac synthase and CMP-Neu5Ac transporter, along with NeuB2,production of Neu5Ac may be enhanced. N-acetylmannosamine (ManNAc)synthesis within a plant may be achieved via several methods. Forexample, ManNAc may be synthesized by expressing within a plant,UDP-GlcNAc 2-epimerase, for example a bacterial UDP-GlcNAc 2-epimerase,which converts UDP-GlcNAc into ManNAc in an irreversible reaction.UDP-GlcNAc is present in the cytosol since it feeds the N-glycanssynthesis pathway. ManNAc synthesis may also be achieved by expressing aGlcNAc-2 epimerase from other sources. Alternatively, ManNAc-6-phosphatemay be formed, followed by hydrolysis with a phosphatase (in transgenicplants). With this approach GlcNAc-6-phosphate 2-epimerase, for examplea bacterial GlcNAc-6-phosphate 2-epimerase, or a mammalian UDP-GlcNAc2-epimerase/ManNAc kinase is expressed within a plant.

To ensure sialylation of N-glycans from cytosolic sialic acid, bacterialor mammalian CMP-Neu5Ac synthase, mammalian CMP-Neu5Ac transporter, amammalian galactosyltransferase, and mammalian sialyltransferase may beco-expressed within a plant (Misaki, R., et. Al. 2006).

The present invention will be further illustrated in the followingexamples.

EXAMPLES Methods

Polyclonal antibodies directed against the synthetic FLAG sequencepolypeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; SEQ ID NO:1) were preparedin rabbits at Eurogentec (Seraing, Belgium). C₁₈ Bond-Elut cartridgeswere from Varian (Sugarland, Tex.). Escherichia coli DH5-alpha andAgrobacterium tumefaciens LBA4404 were used for cloning experiments andtransformation of tobacco cells or M. sativa, respectively. Nicotianatabacum cv Bright Yellow 2 (BY-2) cells were grown as described inGomord et al. (1998).

Cloning of Neu5Ac Lyase and neuB2 Genes and Construction of the PlantExpression Vector

Neu5Ac lyase and neuB2 genes were amplified by PCR. The gene for Neu5Aclyase was amplified from E. coli K1 genomic DNA by PCR using primers:

Lyase-P1: (SEQ ID NO: 6) 5′-AATAGGCCATTACGGCCATGGCAACGAATTTACGTGG-3′ andLyase-P2: (SEQ ID NO: 7) 5′-AATAGGCCGAGGCGGCCTCACCCGCGCTCTTGCAT-3′.For neuB2 gene, the following primers were used to obtain the DNAfragment:

neuB2-P1: (SEQ ID NO: 8) 5′-AATAGGCCATTACGGCCATGAAAAAAACTTTAATC-3′ andneuB2-P2: (SEQ ID NO: 9) 5′-AATAGGCCGAGGCGGCCTTACTCACGGATAAGCTC-3′.These amplified DNAs were placed under the cauliflower mosaic virus(CaMV) 35S promoter of plasmid vector pDNR-LIB for the Neu5Ac lyase geneand binary plasmid vector pCAMBIA 2300 for the neuB2 gene, respectively.The expression cassette of CaMV35S promoter, Neu5Ac lyase, and nopalinesynthase (Nos) terminator was introduced into plant expression vectorpBLTI 121 (Pagny et al., 2000) with kanamycin resistance gene.

To generate the pBLTI Neu5Ac lyase-FLAG and pBLTI neuB2-FLAG plasmids,the following four primers, designed to amplify the genes with the FLAGpeptide encoded at the C-terminal end of the proteins were used:

Lyase-FLAG-P1: (SEQ ID NO: 2)5′-CGGGGTACCAGAGAGATGGCAACGAATTTACGTGGC-3′, Lyase-FLAG-P2:(SEQ ID NO: 3) 5′ GCCGAGCTCTCACTTGTCATCGTCATCCTTGTAATCCATCCCGCGCTCTTGCATCAACTG-3′, neuB2-FLAG-P1: (SEQ ID NO: 4)5′-CGGGGTACCAGAGAGATGAAAAAAACTTTAATCATCGC-3′ and neuB2-FLAG-P2:(SEQ ID NO: 5) 5′-GCCGAGCTCTCACTTGTCATCGTCATCCTTGTAATCCATCTCACGGATAAGCTCATCTTC-3′,

These amplified sequences were generated by PCR with the followingprogram for 30 cycles: denaturation at 94° C. for 1 min, annealing for 1min at 58° C., and polymerization at 72° C. for 3 min. PCR products werecloned into pCR®-BLUNT II-TOPO® (Invitrogen). Before expressing therecombinant proteins in plant cells, we confirmed all of the modifiedcDNA constructs by sequencing. Subsequently, the inserts were digestedwith KpnI and SacI and then cloned into KpnI-SacI-digested pBLTI 121.Each vector (pBLTI 121 or pCAMBIA 2300) was introduced in Agrobacteriumtumefaciens strain LBA4404 via heat shock transformation.

Expression in BY2 Cells

Tobacco BY2 cells were maintained in Murashige and Skoog (1962) mediumand used for transformation. The pBLTI121-derived constructs weretransferred into Agrobacterium (LBA4404) (Hofgen and Willmitzer, 1988).Transgenic Agrobacterium cells were selected on YEB medium containing100 μg·mL⁻¹ kanamycin and used to transform suspension-cultured cells oftobacco as described in Gomord et al. (1998). Transformants wereselected and maintained in MS medium containing antibiotics (kanamycinat 100 μg mL⁻¹ and cefotaxime at 250 μg·mL⁻¹). Genomic DNA and mRNA wereprepared from each transformant, and it was confirmed that objectedgenes were inserted and expressed in tobacco suspension-cultured cellsby PCR and RT-PCR. After immunoscreening, microcalli producing therecombinant proteins were used to initiate suspension cultures oftransgenic cells (Gomord et al., 1998).

Expression in Alfalfa Plants

Alfalfa transformation was performed essentially as described in Tian etal., (Tian et al., 2002) with the following modifications. Alfalfagenotype R2336 was transformed using Agrobacterium tumefaciens AGL1. Theco-culture step was performed with an undiluted culture at 0.8 to 1 OD,and 3% sucrose was used in the Sh2K medium instead of 1.5% sucrose.

Preparation of Cell Extracts for Assay of Neu5Ac Lyase and NeuB2Activity.

One gram of four day old cultures of BY2 suspension-cultured cells oftransformants or 600 mg of fresh leaves of M. sativa were harvested anddisrupted in Solution A (100 mM HCl buffer (pH=7.4) containingproteinase inhibitors (pepstatine 1 μg·mL⁴, E64 1 μg·mL⁻¹ and PMSF 1 mM,Sigma). Cell extracts were then centrifuged at 10000 g for 10 min at 4°C. and proteins were precipitated with ammonium sulfate (finalconcentration 80%) and then dialysed against Solution B (100 mM Tris-HClbuffer, pH=7.4 for Neu5Ac lyase assays or pH=8.5 for NeuB2 assays and 10mM MgCl₂) with Spectra/Por® membrane (cut-off 10000 Da). Proteins werethen utilised for enzymes assays or immunodetections.

Immunodetection of Neu5Ac Lyase-FLAG and NeuB2-FLAG

The proteins were solubilized in a denaturating buffer (20 mM Tris-HClpH 6.8, 0.3% β-mercaptoethanol, 5% (v/v) glycerol and 1% (w/v) SDS),boiled for 5 min and separated by SDS-PAGE in 15% polyacrylamide gels.Proteins were then transferred onto a nitrocellulose membrane. Forimmunodetection, membranes were probed with a rabbit antiserum raisedagainst the FLAG epitope. Proteins were detected by incubation with goatanti-rabbit antibodies conjugated to horseradish peroxidase followed bythe revelation using 4-chloronaphtol or by a chemiluminescence reaction.

Neu5Ac Lyase and Synthase Assays

Soluble enzyme activities were assayed by incubating cell extracts withPEP 4 mM, NADH 4 mM, NaHCO₃ 20 mM and DTE 10 mM. Oxydation of NADH wasmeasured by a diminution of absorbance at 340 nm after 10 min. Lyaseactivity of Neu5Ac lyase was assayed by measuring the formation ofManNAc after incubating transformants cells extracts with Neu5Ac. Cellsextracts were incubated 2 h at 37° C. in Solution B (100 mM Tris-HClbuffer, pH=7.4 and 10 mM MgCl₂) containing proteinase inhibitors(pepstatine 1 μg·mL⁻¹, E64 1 μg·mL¹ and PMSF 1 mM) and Neu5Ac 40 mM.Synthase activity of Neu5Ac lyase was assayed by measuring the formationof Neu5Ac after incubating transformants cells extracts with ManNAc andpyruvate. Cells extracts were incubated 2 h at 37° C. in Solution B (100mM HCl buffer, pH=7.4 and 10 mM MgCl₂) containing proteinase inhibitors(pepstatine 1 μg·mL⁻¹, E64 1 μg·mL¹ and PMSF 1 mM) and ManNAc 20 mM andpyruvate 40 mM. Synthase activity of NeuB2 was assayed by measuring theformation of Neu5Ac after incubating transformants cells extracts withManNAc and PEP. Cells extracts were incubated 2 h at 37° C. in SolutionB (100 mM Tris-HCl buffer, pH=7.4 and 10 mM MgCl₂) containing proteinaseinhibitors (pepstatine 1 μg·mL⁻¹, E64 1 μg·mL⁻¹ and PMSF 1 mM) andManNAc 10 mM and PEP 10 mM. The reactions were stopped by heating 5 minat 80° C. and purified by successive elution with water on a C₁₈Bond-Elut cartridge, lyophilised and derived for GC-EI-MS analysis.

Feeding Experiments

Four day old tobacco BY2 cells were incubated in BY2 medium for two daysat 37° C. with Neu5Ac 10 mM or ManNAc 30 mM to assay Neu5Ac lyase orsynthase activity in vivo respectively. After 2 d, BY2 cells were washedwith BY2 medium without Neu5Ac or ManNAc and harvested. The cells wereheated at 70° C. for 15 min in 70% ethanol to inactivate enzymes andthen ground in a potter homogenizer. The homogenate was washed two timeswith 70% ethanol at 70° C. The remaining pellet and the supernatant wereconsidered as representatives of the cell walls and the cytosolic freemonosaccharides respectively. Monosaccharides of the supernatantfraction were then analysed by gas chromatography

GC Analysis

For the enzymatic assays, the reaction mixtures were first submitted toa purification step on C18 Seppack cartridges. The monosaccharides wereeluted in 100% water. After lyophilisation, the samples were submittedto a 16 h methanolysis at 80° C. with dry 500 μL of 2 M methanolic-HCl.After evaporation of the methanol, the samples were re-acetylated byaddition of 204 of anhydrous acetic anhydride and 20 μL of pyridine. Theresulting N-acetyl methyl glycosides (methyl ester) were dried and thenconverted into their TMS-derivatives and separated by gas chromatography(GC). The gas chromatograph was equipped with a flame ionizationdetector, a WCOT fused silica capillary column (length 25 m, i.d. 0.25mm) with CP-Sil 5 CP as stationary phase and helium as gas vector. Theoven temperature program was: 2 min at 120° C., 10° C./min to 160° C.,and 1.5° C./min to 220° C. and then 20° C./min to 280° C. Thequantification of sugar was done by integration of peaks anddetermination of the corresponding molar values using response factorsestablished with standard monosaccharides.

Transient Expression in Plants

Agrobacterium Growth.

The Agrobacterium clones containing a binary vector bearing the desiredDNA constructs described above were grown for 24 hours at 28° C. in 2 mLof YEB or LB medium containing 25 and 50 μg/mL of carbenicillin andkanamycin, respectively. 10 μL of these cultures were used as startinginoculums to generate cultures of 25 mL of YEB induction medium (YEBmedium, 10 mM 2 (N morpholino) ethanesulfonic acid (MES), pH adjusted to5.6, 25 mg/L carbenicillin, 50 mg/L kanamycin, 20 μM acetosyringone).The latter were grown in rotary shaker (220 rpm) incubator at 28° C. for18 hours or until an optical density at 600 nm (OD600) of 0.8 to 1 wasreached.

Growth of Non-Transgenic Tobacco.

Nicotiana benthamiana and Nicotiana tabacum plants were grown from seedsin a peat-based substrate (AgroMix) in a greenhouse. Seedlings wereinitially raised in a nursery and later transplanted to pots. Plantswere irrigated twice a day and receive 180 ppm of nitrogen at eachapplication. Greenhouse condition were kept at 25° C. during the day and21° C. during the night, under a long day photoperiod regime (16 hlight/8 h dark cycles), with artificial lightning of 20 Watt m⁻² atplant level. Plants can be used at different growth stage, but werepreferentially selected between 5 to 8 weeks of growth.

Transient Expression of Constructs in Tobacco.

Two transient expression methods were used in the present invention:Agro-inoculation or Agro-infiltration. In both methods, a mixture of twoor three Agrobacteria cultures bearing the transfer-DNA (t-DNA) ofinterest are forced to enter into the intercellular spaces of theleaves. Once the physical barrier of the epidermis is crossed, theAgrobacteria infect neighbouring cells transferring t-DNA copies intothe plant cells. With these methods, passage oft-DNA inside the nucleusis transient, the genes present on the t-DNA are episomally transcribedand the mRNA translated, leading to the production of the protein ofinterest in infected cells. The Agro-inoculation technique uses pressureapplied with a syringe to insert the Agrobacteria mixture within theplant tissue, whereas the Agro-infiltration uses a controlled vacuum.

The Agrobacterium culture prepared as described earlier was centrifuged8 min at 10 000 g, resuspended in the same volume of inoculation medium(10 mM MgCl2, 10 mM MES, adjusted to pH 5.6, and supplemented with 100μM acetosyringone) and kept at room temperature (RT, 23° C.) for 1 hprior to inoculation. Alternatively, the suspension can be kept at 4° C.for 24 hours prior to inoculation. Transient transformation of N.benthamiana and N. tabacum were essentially performed as described inLiu and Lomonossoff (2002, Journal of Virological Methods, 105:343-348),with the following modifications. For the expression of the constructsdescribed above, a mixture of two Agrobacteria strains was inoculated.The first strain contained one of the clones described above and thesecond strain contained the HcPro suppressor of silencing from thePotato Virus Y under the control of the 35S promoter. After inoculation,the plants were incubated in a greenhouse. Temperature was kept at aminimum 23° C. during the day and 21° C. during the night. Plants wereirrigated twice a day and received 180 ppm of nitrogen at eachapplication. Harvest of biomass was undertaken after 4-8 days.

Preparation of Soluble Protein Extracts from Transformed Biomass.

Leaves were analyzed directly after harvesting or after freezing thebiomass at −80° C. A biomass of Agro-inoculated or Agro-infiltratedleaves of ˜0.1-1 g was weighted and used to generate a total proteinliquid extract.

Several extraction methods were used to generate total protein extracts:by grounding the vegetable tissue with a mortar and a pestle, by using apolytron, or by pulverizing it in a MixerMill300 (MM300) from Retsch.0.1-1 g of plant biomass was transferred into a clean and pre-cooledmortar. Cold extraction buffer (Tris 50 mM, NaCl 150 mM pH 7.4 buffercontaining, 2 mM CaCl₂ and 4% butanol) was added at a 1:3 ration (w/v)as well as PMSF and chymostatin to final concentrations of 1 mM and 10μM, respectively. Leaves were ground with a pestle until a homogeneouspreparation was obtained. The plant extract was then transferred into a1.5 mL microtube and centrifuged at 20,000 g for 20 min at 4° C.Alternatively, 0.1 g of plant tissue with 0.3 mL of extraction bufferwas introduced into non-sterile 1.5 microtube. A tungsten bead was addedto each tube. The box was submitted to 3 min cycle of agitation at 30Hz. The cycle was repeated 2 times. The plant extracts were thencentrifuged as described above. Alternatively, 1 g of biomass waspulverized with 3 mL of extraction buffer using a polytron.

Following centrifugation, the supernatant was transferred into a cleanmicrotube and maintained on ice. Finally, the total protein content ofindividual protein extracts was measured by the Bradford method usingBSA as the reference protein.

Example 1 Expression of the E. coli Neu5Ac Lyase in Tobacco BY2 Cells

The gene encoding Neu5Ac lyase from Escherichia coli K1 (accessionnumber: D00067) was introduced into tobacco BY2 cells. Transgenic BY2calli were generated after Agrobacterium mediated transformation withthe plasmid pBLTI121 containing the E. coli K1 Neu5Ac lyase gene.Another construct was tagged at its C-terminus with a FLAG epitope toallow immunodetection of the recombinant protein in transformants. Thetransformants selected for kanamicin resistance were analysed for mRNAlevels by RT-PCR. Thirty-six from 48 transformants expressing the Neu5Aclyase transcript and 30 from 50 transformants expressing the Neu5Aclyase-FLAG transcript were obtained. Calli harbouring the highest mRNAexpression levels were transferred in suspension cultures for thecharacterisation of Neu5Ac lyase activity. The presence of the Neu5Aclyase-FLAG was determined in protein cytosolic extracts of transformedBY2 cells by western-blot analysis. A single protein band with anapparent MW of 32 kDa was specifically immunodetected in the transformedcells using anti-FLAG antibodies (FIG. 1a ).

Enzymatic assays were carried out on soluble protein extracts fromsuspension-cultured BY2 cells expressing the Neu5Ac lyase and Neu5Aclyase-FLAG. Both extracts showed a lyase activity. Further analysis wasconducted on protein extracts isolated from cells expressing thenon-tagged lyase. These extracts were first incubated in the presence ofNeu5Ac to investigate their lyase activity. FIGS. 1b and 1c show the GCprofiles of the end-products formed by incubating a Neu5Ac lyase proteinextract in absence (FIG. 1b ) or presence of Neu5Ac (FIG. 1c ) at pH 7and at 37° C. Three signals (peak 1, is a shoulder of an endogenoussignal) were clearly detected when the extract was incubated withNeu5Ac. These signals eluted at retention times similar to those ofpyranose (peak 1) and furanose (peaks 2 and 3) forms of standard ManNAc.Gas chromatography coupled to Electron Impact Mass Spectrometry (GC-EIMS) of the sample confirmed the assignment of these signals to1-O-methyl persilyl derivatives of ManNAcp (FIG. 1e ) and ManNAcf (FIG.1f ). Diagnostic ions at m/z=173 and 186 were assigned to fragmentscontaining the nitrogen atom, as usually observed for aminosugars.

Incubation of a cytosolic protein extract from wild-type tobacco BY2cells with Neu5Ac in similar conditions did not result in any formationof ManNAc (data not shown), thus demonstrating the absence of anendogenous lyase activity in plants. This data indicates that BY2 cellstransformed with the E. coli Neu5Ac lyase gene expressed a functionalenzyme able to cleave Neu5Ac into D-ManNAc.

The optimum pH of the recombinant enzyme was determined to be about 7,based upon GC quantification of D-ManNAc generated in assays carried outin a 4-10 pH range (data not shown). Furthermore, the recombinant enzymeexhibited a temperature dependent activity with high lyase activityobserved in a 25-37° C. range. At pH 7 and at 37° C., a soluble proteinextract from transformed cells formed 0.5 μmole of ManNAc from 10 μmolesof Neu5Ac, in 1 h. Below 15° C., only residual activity was detected.

The ability of the recombinant Neu5Ac lyase to synthesise Neu5Ac wasdetermined by incubating protein extracts of transformed tobacco BY2cells with D-ManNAc and pyruvate at pH 7 and 37° C. FIGS. 2a and 2b showthe GC profiles of end-products after incubation in the absence orpresence of substrates respectively. When compared to the controlprofile (FIG. 2a ), GC profile of the reaction conducted in the presenceof D-ManNAc and pyruvate showed a signal at a retention time expectedfor Neu5Ac (box in FIG. 2b ). The electron impact mass spectrum (EI MS)of this signal (FIG. 2c ), exhibited the diagnostic ions at m/z=298 and420 specific for Neu5Ac fragmentation, as well as the ion at m/z=186assigned to the nitrogen-containing fragment. This data indicates thatthe recombinant lyase is able to synthesise Neu5Ac in presence ofD-ManNAc and pyruvate.

In planta activity of the Neu5Ac lyase was determined by feeding tobaccoBY2 cells with 10 mM Neu5Ac. The toxicity of Neu5Ac on tobacco BY2 cellswere investigated, and no toxic effects were observed over a 48 h periodby testing the cell viability using propidium iodide and fluoresceindiacetate. The formation of D-ManNAc was determined by analysingcytosolic monosaccharides by GC after a 48 h period at temperatureranging from 23° C. to 37° C. D-ManNAc was detected in all treatments(FIG. 1d ). The quantification of D-ManNAc by GC showed a 25-foldincrease in the content of this aminosugar at 37° C. compared to 23° C.These in vivo experiments demonstrate that the Neu5Ac lyase isbiologically active in planta and is able to act upon an exogenouslysupplied substrate.

Expression of Campylobacter jejuni NeuB2 in Tobacco BY2 and AlfalfaPlants

Neu5Ac synthase, NeuB2, from Campylobacter jejuni (accession number:NC002163) catalyzes the formation of Neu5Ac by condensation of D-ManNAcand PEP. Transgenic BY2 calli were generated after Agrobacteriummediated transformation with the plasmid pBLTI121 containing the neuB2cDNA. For immunodetection of the protein, a second construct was taggedat its C-terminus end with a FLAG epitope. The transformants selectedfor kanamicin resistance were analysed for mRNA levels by RT-PCR. Calliharbouring the highest mRNA expression levels were transferred insuspension cultures for analysis. The accumulation of NeuB intransformed BY2 cells was then determined by western-blot analysis of aprotein soluble extract isolated from BY2 cells transformed with theNeuB2-FLAG sequence. As illustrated in FIG. 3a , anti-FLAG antibodiesspecifically recognised a single protein band at MW-37 kDa consistentwith the expected molecular weight of the synthase. neuB2 was alsointroduced in alfalfa plants by Agrobacterium-mediated transformationand in vitro regeneration of plants (Tian et al., 2002). From 34transformed plants, 29 were demonstrated to express the neuB2transcript.

Prior to the analysis of transformed cells and plants expressing thebacterial Neu5Ac synthase, the occurrence of endogenous Neu5Ac synthaseactivity was investigated. Protein soluble extracts from both wild-typetobacco BY2 cells and alfalfa plants were incubated with D-ManNAc andPEP. The monosaccharides formed in the assays were separated by GC andcharacterised by GC-EI MS. No peak or EI MS diagnostic ions assigned toNeu5Ac were detected, indicating that plants do not express endogenousenzymes able to form Neu5Ac by condensation of PEP onto D-ManNAc.

The synthase activity of the recombinant NeuB2 expressed in plants wasdetermined by incubation of D-ManNAc and PEP with soluble proteinextracts isolated from tobacco BY2 cells or alfalfa plants transformedwith neuB2 gene. FIGS. 3b and 3c show the GC profiles obtained byincubation of a transformed alfalfa extract without (FIG. 3b ) or with(FIG. 3c ) D-ManNAc and PEP at pH=8 and 37° C. A peak eluted at theexpected retention times for Neu5Ac was specifically detected afterincubation with the substrates of the synthase. EI MS of this peakexhibited a fragmentation pattern similar to the one of a standardNeu5Ac, with diagnostic ions at m/z=298 and 420. Those ions were notdetected in the EI-MS spectrum of the corresponding region of the GCprofile after incubation in absence of D-ManNAc (FIG. 3b ).

The same result was obtained by the analysis of tobacco BY2 cellsexpressing the NeuB2 or the NeuB2-FLAG sequence.

Therefore, expression of neuB2 in both tobacco BY2 cells and alfalfaplants results in the production of a functional Neu5Ac synthase.

REFERENCES

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All citations are hereby incorporated by reference.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

What is claimed is:
 1. A method of synthesizing sialic acid comprising,i) providing a transgenic plant comprising a nucleotide sequenceencoding bacterial N-acetyl neuraminic acids lyase utilizingN-acetyl-D-mannosamine as a substrate, the nucleotide sequenceoperatively linked with a regulatory region that is active in the plant,and ii) growing the transgenic plant and expressing the nucleotidesequence thereby synthesizing sialic acid; and iii) recovering freesialic acid from the transgenic plant.
 2. A method of synthesizingsialic acid comprising, i) providing a transgenic plant cell comprisinga nucleotide sequence encoding bacterial N-acetyl neuraminic acid lyaseutilizing N-acetyl-D-mannosamine as a substrate, the nucleotide sequenceoperatively linked with a regulatory region that is active in the plant,and ii) growing the transgenic plant cell and expressing the nucleotidesequence thereby synthesizing sialic acid; and iii) recovering freeN-acetyl neuraminic acid (Neu5Ac) from the transgenic plant cell.
 3. Themethod of claim 1, wherein the regulatory region is selected from thegroup consisting of a constitutive promoter, an inducible promoter, atissue specific promoter, and a developmental promoter.